This innovative proposal focuses on the fundamental question of T-cell receptor (TCR) signal initiation by peptide-MHC ligands (pMHCs). A unique feature of TCR triggering is that while soluble pMHC is incapable of triggering in solution even at very high concentrations, pMHCs on antigen presenting cells (APCs) trigger TCR and activate T-cells with high potency. Using artificial antigen presentation systems with defined components, we determined that TCR can be triggered by very few (1 to 10) monomeric agonist pMHCs anchored on a surface, independent of endogenous pMHCs or other molecules on the surface of real APCs. In addition to surface-anchoring, the triggering also critically depends upon active T-cell adhesion and intact cytoskeletal function. Considering the sufficiency of these three components in TCR triggering, the dynamic nature of T cell-APC interaction, and the constant mechanical stresses on pMHC-TCR interaction, we propose a new model for TCR triggering, the receptor deformation model. We hypothesize that TCR signaling is initiated by conformational change of the TCR/CD3 complex induced by a pulling force originating from the dynamic T-cell cytoskeleton, and transmitted through pMHC-TCR interactions with sufficient resistance to rupture under force. This model not only offers a straightforward mechanism for TCR signal initiation, but also explains the extraordinary sensitivity and specificity of TCR triggering. We will test our hypothesis from two perspectives. In Specific Aim 1, we will define the physical and mechanical properties of a surface that confers pMHC with potent TCR triggering capacity. We hypothesize that a relatively immobile, stiff, and moderately adhesive surface for pMHC anchoring will enable transmission of cytoskeletal force to TCR through pMHC-TCR binding most efficiently. In Specific Aim 2, we will directly test the role of mechanical force in TCR triggering using single molecule research design. External mechanical forces will be exerted on the TCR to test their effect on triggering. The extensibility (conformational change under force) of TCR will be determined using atomic force microscopy (AFM). Finally, the rupture force (a parameter of binding strength under force) of pMHC-TCR binding will be measured using AFM, to test our hypothesis that this determines pMHC potency. In our view, by incorporating the omitted dynamic aspect of the 2D interaction between TCR and pMHC, this model represents a new step in the evolution of TCR triggering theory, from the focus on affinity to 3D kinetics, to 2D kinetics, to our dynamic 2D kinetics. The introduction of an external force to pMHC-TCR binding provides a new dimension to research on immune receptor biology. PROJECT NARRATIVE: As the very first step of antigen recognition by T-cells, TCR triggering is a critical event in the adaptive immune response. Here, we propose a new model for TCR triggering, the receptor deformation model, which explains the extraordinary sensitivity and specificity of TCR triggering, and test this model using biophysical methods and single molecule research design. An understanding of the mechanism of TCR triggering has profound implications in developing new approaches for enhancing or subduing immune responses to treat human disease.